Insufficient stem cell engraftment is a key problem that limits the application of transplant therapies for genetic and malignant diseases involving the hematopoietic system. Experimental studies have shown that depletion of primitive stem cells in the bone marrow is often required in the host before long- term engraftment from donor stem cells is achieved. In current clinical transplant protocols, this is usually accomplished using recipient conditioning with aggressive doses of whole body irradiation or busulfan. These treatments are limited by harsh side-effects. Recipient treatment with an agent capable of selectively depleting stem cells in the bone marrow microenvironment has the promise of providing less toxic and more specific replacement. Our goal in this phase I proposal is to demonstrate the proof of concept that these toxic treatments can be substituted by an agent that is specifically directed against the true hematopoietic stem cells (HSC) of the recipient by virtue of its existence in low oxygen tension. Such a strategy would allow the use of milder conditioning therapy in maximizing the engraftment of stem cells corrected by gene therapy in autologous transplants. Specifically, the initial development will comprise of administering tirapazamine (TPZ) and a closely related TPZ analog (SN 30000). We have already identified TPZ as being capable of depleting hypoxic bone marrow HSCs in the host and where we expect to achieve robust and persistent donor-type engraftment in murine recipients of syngeneic bone marrow. It is anticipated that we will identify an improved HSC transplant conditioning regimen that, together with the safe and effective means of delivering a gene therapy, can be extended to large animal models and eventually to human clinical application. Specific Aims: I. Optimize the bone marrow stem cell depleting properties of the hypoxia-selective cytotoxin tirapazamine (TPZ) and SN 30000; II. Assess the ability of these drugs to provide for long-term engraftment of normal donor stem cells in a bone marrow transplant model.7. Project Narrative Insufficient stem cell engraftment is a key problem that limits the application of transplant therapies for genetic and malignant diseases involving the hematopoietic system. Recipient treatment with an agent capable of selectively depleting stem cells in the bone marrow microenvironment has the promise of providing less toxic and more specific replacement for either irradiation or busulfan in stem cell-corrective gene therapy. We also expect that development of such a method will accelerate the clinical application of gene therapy directed at permanently correcting ?-thalassemia (Cooley's anemia) and sickle cell anemia which constitute some of most common and serious of the genetic diseases. [unreadable] [unreadable] [unreadable]